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Updated: Jul 14, 2026
Glutamine Flux Imaging Using Genetically Encoded Sensors
Published on: July 31, 2014
A model for glutathione binding and activation in the fosfomycin resistance protein, FosA
Rachel E Rigsby1, Daniel W Brown, Eric Dawson
1Department of Chemistry and The Vanderbilt Institute of Chemical Biology, Vanderbilt University, Nashville, TN 37232, USA.
Abstract:
The genomically encoded fosfomycin resistance protein from Pseudomonas aeruginosa (FosA(PA)) utilizes Mn(II) and K(+) to catalyze the addition of glutathione (GSH) to C1 of the antibiotic rendering it inactive. Although this protein has been structurally and kinetically characterized with respect to the substrate, fosfomycin, questions remain regarding how the enzyme binds the thiol substrate, GSH. Computational studies have revealed a potential GSH binding site in FosA(PA) that involves six electrostatic or hydrogen-bonding interactions with protein side-chains as well as six additional residues that contribute van der Waals interactions. A strategically placed tyrosine residue, Y39, appears to be involved in the ionization of GSH during catalysis. The Y39F mutant exhibits a 13-fold reduction of catalytic activity (k(cat)=14+/-2s(-1)), suggesting a role in the ionization of GSH. Mutation of five other residues (W34, Q36, S50, K90, and R93) implicated in ionic of hydrogen-bonding interactions resulted in enzymes with reduced catalytic efficiency, affinity for GSH, or both. The mutant enzymes were also found to be less effective resistant proteins in the biological context of Escherichia coli. The more conservative W34H mutant has native-like catalytic efficiency suggesting that the imidazole NH group can replace the indole group of W34 that is important for GSH binding. In the absence of co-crystal structural data with the thiol substrate, these results provide important insights into the role of GSH in catalysis.
Insights
The Pseudomonas aeruginosa fosfomycin resistance protein (FosA(PA)) inactivates fosfomycin by adding glutathione (GSH). This study identifies key protein residues essential for binding and catalyzing the glutathione reaction, crucial for antibiotic resistance.
Area of Science:
- Biochemistry
- Molecular Biology
- Enzymology
Background:
- Fosfomycin resistance in Pseudomonas aeruginosa is mediated by the FosA(PA) protein.
- FosA(PA) catalyzes the glutathione S-transferase reaction, adding glutathione (GSH) to fosfomycin.
- The precise mechanism of GSH binding and activation by FosA(PA) remains incompletely understood.
Purpose of the Study:
- To elucidate the role of specific amino acid residues in the binding and catalytic mechanism of GSH by FosA(PA).
- To investigate the contribution of electrostatic, hydrogen-bonding, and van der Waals interactions in GSH substrate recognition.
- To understand the structural basis of fosfomycin resistance conferred by FosA(PA).
Main Methods:
- Site-directed mutagenesis of key residues within FosA(PA).
- Enzymatic assays to measure catalytic activity (kcat) and substrate affinity (Km) of wild-type and mutant proteins.
- Assessment of fosfomycin resistance in Escherichia coli expressing mutant FosA(PA) proteins.
Main Results:
- Mutation of tyrosine 39 (Y39F) significantly reduced catalytic activity, indicating its role in GSH ionization.
- Mutations in residues W34, Q36, S50, K90, and R93 impaired catalytic efficiency and/or GSH binding affinity.
- The W34H mutant retained near-native catalytic efficiency, suggesting imidazole NH can substitute for indole in GSH binding.
- Mutant enzymes conferred reduced fosfomycin resistance in vivo.
Conclusions:
- Specific residues, including Y39 and W34, are critical for the efficient binding and catalysis of GSH by FosA(PA).
- Electrostatic and hydrogen-bonding interactions play a significant role in recognizing the GSH substrate.
- These findings provide crucial insights into the mechanism of fosfomycin resistance mediated by FosA(PA).
